Antibodies against human csf-1r and uses thereof

ABSTRACT

The present invention relates to antibodies against human CSF-1R (CSF-1R antibody), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

The present invention relates to antibodies against human (CSF-1R(CSF-1R antibody), methods for their production, pharmaceuticalcompositions containing said antibodies, and uses thereof.

BACKGROUND OF THE INVENTION

The CSF-1 receptor (CSF-1R; synonyms: M-CSF receptor; Macrophagecolony-stimulating factor 1 receptor, EC 2.7.10.1, Fms proto-oncogene,c-fms, Swiss Prot P07333, CD115, (SEQ ID NO: 23)) is known since 1986(Coussens, L., et al., Nature 320 (1986) 277-280). CSF-1R is a growthfactor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth,P, and Stanley, E. R., Curr. Top. Microbiol. Immunol. 181 (1992)141-67).

CSF-1R is the receptor for M-CSF (macrophage colony stimulating factor,also called CSF-1) and mediates the biological effects of this cytokine(Sherr, C. J., et al., Cell 41 (1985) 665-676). The cloning of thecolony stimulating factor-1 receptor (also called c-fms) was describedfor the first time in Roussel, M. F., et al., Nature 325 (1987) 549-552.In that publication, it was shown that CSF-1R had transforming potentialdependent on changes in the C-terminal tail of the protein including theloss of the inhibitory tyrosine 969 phosphorylation which binds Cbl andthereby regulates receptor down regulation (Lee, P. S., et al., Embo J.18 (1999) 3616-3628).

CSF-1R is a single chain, transmembrane receptor tyrosine kinase (RTK)and a member of the family of immunoglobulin (Ig) motif containing RTKscharacterized by repeated Ig domains in the extracellular portion of thereceptor. The intracellular protein tyrosine kinase domain isinterrupted by a unique insert domain that is also present in the otherrelated RTK class III family members that include the platelet derivedgrowth factor receptors (PDGFR), stem cell growth factor receptor(c-Kit) and fins-like cytokine receptor (FLT3). In spite of thestructural homology among this family of growth factor receptors, theyhave distinct tissue-specific functions. CSF-1R is mainly expressed oncells of the monocytic lineage and in the female reproductive tract andplacenta. In addition expression of CSF-1R has been reported inLangerhans cells in skin, a subset of smooth muscle cells (Inaba, T., etal., J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, A. M., etal., Oncogene 8 (1993) 371-378) and microglia (Sawada, M., et al., BrainRes, 509 (1990) 119-124).

The main biological effects of CSF-1R signaling are the differentiation,proliferation, migration, and survival of hematopoietic precursor cellsto the macrophage lineage (including osteoclast). Activation of CSF-1Ris mediated by its ligand, M-CSF. Binding of M-CSF to CSF-1R induces theformation of homodimers and activation of the kinase by tyrosinephosphorylation (Stanley, E. R., et al., Mol. Reprod. Dev. 46 (1997)4-10). Further signaling is mediated by the p85 subunit of PI3K and Grb2connecting to the PI3K/AKT and Ras/MAPK pathways, respectively. Thesetwo important signaling pathways can regulate proliferation, survivaland apoptosis. Other signaling molecules that bind the phosphorylatedintracellular domain of CSF-1R include STAT1, STAT3, PLCy, and Cbl(Bourette, R. P. and Rohrschneider, L. R., Growth Factors 17 (2000)155-166).

CSF-1R signaling has a physiological role in immune responses, in boneremodeling and in the reproductive system. The knockout animals foreither M-CSF-1 (Pollard, J. W., Mol. Reprod. Dev. 46 (1997) 54-61) orCSF-1R (Dai, X. M., et al., Blood 99 (2002) 111-120) have been shown tohave osteopetrotic, hematopoietic, tissue macrophage, and reproductivephenotypes consistent with a role for CSF-1R in the respective celltypes.

Sherr, C. J. et al., Blood 73 (1989) 1786-1793 relates to someantibodies against CSF-JR that inhibit the CSF-1 activity (see Sherr, C.J., et al., Blood 73 (1989) 1786-1793). Ashum, R. A., et al., Blood 73(1989) 827-837 relates to CSF-1R antibodies. Lenda, D., et al., Journalof immunology 170 (2003) 3254-3262 relates to reduced macrophagerecruitment, proliferation, and activation in CSF-1-deficient miceresults in decreased tubular apoptosis during renal inflammation.Kitaura, H., et al., Journal of dental research 87 (2008) 396-400 refersto an anti-CSF-1 antibody which inhibits orthodontic tooth movement. WO2001/030381 mentions CSF-1 activity inhibitors including antisensenucleotides and antibodies while disclosing only CSF-1 antisensenucleotides. WO 2004/045532 relates to metastases and bone lossprevention and treatment of metastatic cancer by a M-CSF antagonistdisclosing as antagonist anti-CSF-1-antibodies only. WO 2005/046657relates to the treatment of inflammatory bowel disease byanti-CSF-1-antibodies. US 2002/0141994 relates to inhibitors of colonystimulating factors. WO 2006/096489 relates to the treatment ofrheumatoid arthritis by anti-CSF-1-antibodies.

SUMMARY OF THE INVENTION

The invention comprises in one aspect an antibody binding to humanCSF-1R, characterized in binding to the same epitope as the depositedantibody DSM ACC2920 for use in the inhibition of cell proliferation inCSF-1 ligand-dependent and/or CSF-1 ligand-independent CSF-1-Rexpressing cells.

In one embodiment the CSF-1-R expressing cells is a cancer cell.

-   In one embodiment the antibody is characterized in comprising as    heavy chain variable domain CDR3 region a CDR3 region of SEQ ID NO:    1, or SEQ ID) NO: 9.

In one embodiment the antibody is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO:2, and a CDR1 region of        SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO:4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14; or    -   c) a CDR grafted, humanized or T cell epitope depleted antibody        variant of the antibodies of a), or b).

-   Thus the antibodies according to the invention binding to the same    epitope were able to inhibit cell proliferation in CSF-1    ligand-dependent and CSF-1 ligand independent cells.

-   The invention comprises in another aspect an antibody binding to    human CSF-1R, characterized in binding to the same epitope as the    deposited antibody DSM ACC2920.

-   The invention comprises in another aspect an antibody binding to    human CSF-1R, characterized in binding to the same epitope as the    deposited antibody DSM ACC2920;    -   wherein the binding to the same epitope is measured at 25° C. by        Surface Plasmon Resonance (SPR) in an in vitro competitive        binding inhibition assay; and    -   wherein the antibody has the following properties:    -   a) inhibition of the growth of NIH3T3—wildtype CSF-1R        recombinant cells by 90% or more at an antibody concentration of        10 μg/ml; and    -   b) inhibition of the growth of NIH3T3—mutant CSF-1R L301S Y969F        recombinant cells by 60% or more at an antibody concentration of        10 μg/ml.

-   In one embodiment the antibody is characterized in comprising as    heavy chain variable domain CDR3 region a CDR3 region of SEQ ID NO:    1, or SEQ ID NO: 9.

In one embodiment the antibody is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region        of SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14; or    -   c) a CDR grafted, humanized or T cell epitope depleted antibody        variant of the antibodies of a), or b).

-   In one embodiment the antibody binding to human CSF-1R and being    characterized by the above mentioned amino acid sequences and amino    acid sequence fragments is of human IgG1 subclass or is of human    IgG4 subclass.

A further embodiment of the invention is a pharmaceutical compositioncomprising an antibody according to the invention.

-   The invention further comprises a pharmaceutical composition    characterized in comprising the antibody binding to human CSF-1R    being characterized by the above mentioned epitope binding    properties or alternatively by the above mentioned amino acid    sequences and amino acid sequence fragments.-   The invention further comprises the use an of an antibody    characterized in comprising the antibody binding to human CSF-1R    being characterized by the above mentioned epitope binding    properties or alternatively by the above mentioned amino acid    sequences and amino acid sequence fragments for the manufacture of a    pharmaceutical composition.-   The invention further comprises the use of an antibody characterized    in comprising the antibody binding to human CSF-1R being    characterized by the above mentioned epitope binding properties or    alternatively by the above mentioned amino acid sequences and amino    acid sequence fragments for the treatment of a CSF-1R mediated    diseases.-   The invention further comprises the use of an antibody characterized    in comprising the antibody binding to human CSF-1R being    characterized by the above mentioned epitope binding properties or    alternatively by the above mentioned amino acid sequences and amino    acid sequence fragments for the treatment of cancer.-   The invention further comprises the use of an antibody characterized    in comprising the antibody binding to human CSF-1R being    characterized by the above mentioned epitope binding properties or    alternatively by the above mentioned amino acid sequences and amino    acid sequence fragments for the treatment of bone loss.-   The invention further comprises the of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for the prevention or treatment of metastasis.-   The invention further comprises the of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for treatment of inflammatory diseases.

The antibody binds to human CSF-1R preferably with an affinity of atleast 10⁻⁸ mol/l to 10⁻¹² mol/l.

Preferably the antibody is a humanized or human antibody.

A further embodiment of the invention is a nucleic acid encoding a heavychain variable domain and/or a light chain variable domain of anantibody according to the invention. Preferably the nucleic acid encodesa heavy chain of an antibody binding to human CSF-1R, characterized incomprising as heavy chain CDR3 region a CDR3 region of SEQ ID NO: 1, SEQID NO: 9, or SEQ ID NO: 17.

A further embodiment of the invention is a nucleic acid encoding anantibody according to the invention characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region        of SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14; or    -   c) a CDR grafted, humanized or T cell epitope depleted antibody        variant of the antibodies of a), or b).

The invention further provides expression vectors containing nucleicacid according to the invention capable of expressing said nucleic acidin a prokaryotic or eukaryotic host cell, and host cells containing suchvectors for the recombinant production of such an antibody.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of arecombinant human or humanized antibody according to the invention,characterized by expressing a nucleic acid according to the invention ina prokaryotic or eukaryotic host cell and recovering said antibody fromsaid cell or the cell culture supernatant. The invention furthercomprises the antibody obtainable by such a recombinant method.

Antibodies according to the invention show benefits for patients in needof a CSF-1R targeting therapy. The antibodies according to the inventionhave new and inventive properties causing a benefit for a patientsuffering from a tumor disease, especially suffering from cancer.

The invention further provides a method for treating a patient sufferingfrom cancer, comprising administering to a patient diagnosed as havingsuch a disease (and therefore being in need of such a therapy) aneffective amount of an antibody binding to human CSF-1R according to theinvention. The antibody is administered preferably in a pharmaceuticalcomposition.

A further embodiment of the invention is a method for the treatment of apatient suffering from cancer characterized by administering to thepatient an antibody according to the invention.

The invention further comprises the use of an antibody according to theinvention for the treatment of a patient suffering from cancer and forthe manufacture of a pharmaceutical composition according to theinvention. In addition, the invention comprises a method for themanufacture of a pharmaceutical composition according to the invention.

The invention further comprises a pharmaceutical composition comprisingan antibody according to the invention, optionally together with abuffer and/or an adjuvant useful for the formulation of antibodies forpharmaceutical purposes.

The invention further provides pharmaceutical compositions comprising anantibody according to the invention in a pharmaceutically acceptablecarrier. In one embodiment, the pharmaceutical composition may beincluded in an article of manufacture or kit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Growth inhibition of BeWo tumor cells in 3D culture undertreatment with different anti-CSF-1R monoclonal antibodies at aconcentration of 10 μg/ml.

-   -   X axis: viability mean relative light units (RLU) corresponding        to the ATP-content of the cells (CellTiterGlo assay).    -   Y axis: tested probes: Minimal Medium (0.5% FBS), mouse IgG1        (mIgG1, 10 μg/ml), mouse IgG2a (mIgG2a 10 μg/ml), CSF-1 only,        <CSF-1R>9D11.2E8, <CSF-1R>10H2.2F12, and SC-02, clone 2-4A5.    -   Highest inhibition of CSF-1 induced growth was observed with the        anti-CSF-1R antibodies according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention further comprises an antibody binding to human CSF-1R,characterized in comprising as heavy chain variable domain CDR3 region aCDR3 region of SEQ ID NO: 1, or SEQ ID NO: 9.

The invention further comprises said antibody, characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region        of SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14; or    -   c) a CDR grafted, humanized or T cell epitope depleted antibody        variant of the antibodies of a), or b).

The term “antibody” encompasses the various forms of antibodiesincluding but not being limited to whole antibodies, antibody fragments,humanized antibodies, chimeric antibodies, T cell epitope depletedantibodies, and further genetically engineered antibodies as long as thecharacteristic properties according to the invention are retained.

“Antibody fragments” comprise a portion of a full length antibody,preferably the variable domain thereof, or at least the antigen bindingsite thereof. Examples of antibody fragments include diabodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. scFv antibodies are, e.g., described inHouston, J. S., Methods in Enzymol. 203 (1991) 46-88). In addition,antibody fragments comprise single chain polypeptides having thecharacteristics of a V_(H) domain binding to CSF-1R, namely being ableto assemble together with a V_(L) domain, or of a V_(L) domain bindingto CSF-1R, namely being able to assemble together with a V_(H) domain toa functional antigen binding site and thereby providing the property.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from mouse and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a mouse variable region and a human constant region areespecially preferred. Such rat/human chimeric antibodies are the productof expressed immunoglobulin genes comprising DNA segments encoding ratimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of “chimeric antibodies”encompassed by the present invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L., et al., Proc.Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 andU.S. Pat. No. 5,204,244.

The term “CDR-grafted variant” as used within the current applicationdenotes a variable domain of an antibody comprising complementarydetermining regions (CDRs or hypervariable regions) from one source orspecies and framework regions (FRs) from a different source or species,usually prepared by recombinant DNA techniques. CDR-grafted variants ofvariable domains comprising murine CDRs and a human FRs are preferred.

The term “T-cell epitope depleted variant” as used within the currentapplication denotes a variable domain of an antibody which was modifiedto remove or reduce immunogenicity by removing human T-cell epitopes(peptide sequences within the variable domains with the capacity to bindto MHC Class II molecules). By this method interactions between aminoacid side chains of the variable domain and specific binding pocketswith the MHC class ii binding groove are identified. The identifiedimmunogenic regions are mutated to eliminate immunogenicity. Suchmethods are described in general in, e.g., WO 98/52976.

The term “humanized variant” as used within the current applicationdenotes a variable domain of an antibody, which is reconstituted fromthe complementarity determining regions (CDRs) of non-human origin. e.g.from a non-human species, and from the framework regions (FRs) of humanorigin, and which has been further modified in order to alsoreconstitute or improve the binding affinity and specificity of theoriginal non-human variable domain. Such humanized variants are usuallyprepared by recombinant DNA techniques. The reconstitution of theaffinity and specificity of the parent non-human variable domain is thecritical step, for which different methods are currently used. In onemethod it is determined whether it is beneficial to introduce mutations,so called backmutations, in the non-human CDRs as well as in the humanFRs. The suited positions for such backmutations can be identified e.g.by sequence or homology analysis, by choosing the human framework (fixedframeworks approach; homology matching or best-fit), by using consensussequences, by selecting FRs from several different human mAbs, or byreplacing non-human residues on the three dimensional surface with themost common residues found in human mAbs (“resurfacing” or “veneering”).

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-CSF-1Rantibody can be preferably replaced with another amino acid residue fromthe same side chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al., Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

The CSF-1 receptor (CSF-1; synonyms; M-CSF receptor; Macrophagecolony-stimulating factor 1 receptor, EC 2.7.10.1, Fms proto-oncogene,c-fms, Swiss Prot P07333, CD115, (SEQ ID NO: 23)) is known since 1986(Coussens. L. et al., Nature 320 (1986) 277-280). CSF-1R is a growthfactor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth,P., and Stanley, E. R., Curr. Top. Microbiol. Immunol. 181 (1992)141-67).

CSF-1R is the receptor for M-CSF (macrophage colony stimulating factor,also called CSF-1) and mediates the biological effects of this cytokine(Sherr, C J., et al., Cell 41 (1985) 665-676). The cloning of the colonystimulating factor-1 receptor (also called c-fms) was described for thefirst time in Roussel. M. F., et al., Nature 325 (1987) 549-552. In thatpublication, it was shown that CSF-1R had transforming potentialdependent on changes in the C-terminal tail of the protein including theloss of the inhibitory tyrosine 969 phosphorylation which binds Cbl andthereby regulates receptor down regulation (Lee, P. S., et al., Embo J.18 (1999) 3616-3628).

As used herein, “binding to human CSF-1R” refers to an antibodyspecifically binding to the human CSF-1R antigen. The binding affinityis of KD-value of 1.0×10⁻⁸ mol/l or lower at 35° C., preferably of aKD-value of 1.0×10⁻⁹ mol or lower at 35° C. The binding affinity isdetermined with a standard binding assay at 35° C., such as surfaceplasmon resonance technique (Biacore®) (see Example 4).

The term “epitope” denotes a protein determinant capable of specificallybinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually epitopes have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Preferably an antibody according to the inventionbinds specifically to native but not to denatured CSF-JR.

The term “binding to the same epitope as the deposited antibody DSMACC2920” as used herein refers to an anti-CSF-1R antibody of theinvention that binds to the same epitope on CSF-1R to which the antibody<CSF-1R>9D11.2E8 (deposit no. DSM ACC2920) binds. The epitope bindingproperty of an anti-CSF-1R antibody of the present invention may bedetermined using techniques known in the art. The CSF-1R antibody ismeasured at 25° C. by Surface Plasmon Resonance (SPR) in an in vitrocompetitive binding inhibition assay to determine the ability of thetest antibody to inhibit binding of antibody <CSF-1R>9D11.2E8 (depositno. DSM ACC2920) to CSF-1R. This can be investigated by a BIAcore assay(Pharmacia Biosensor AB, Uppsala, Sweden) as e.g. in Example 5. InExample 5 the percentage (%) of expected binding response of the CSF-1Rantibody of the invention competing with the bound the antibody<CSF-1R>9D11.2E8 (deposit no. DSM ACC2920) is calculated by“100*relative Response(general_stability_early)/rMax”, where rMax iscalculated by “relative Response(general_stability_late)*antibodymolecular weight/antigen molecular weight” as described in BIAcore assayepitope mapping instructions. A minimal binding response is alsocalculated from the pairs of identical antibody 1 and 2 (see Example 5).Thereof the obtained maximal value+50% is set as threshold forsignificant competition and thus significant binding to the same epitope(see Example 5 for antibody <CSF-1R>9D11.2E8 calculated threshold is8+4=12). Thus an antibody binding to human CSF-1R, characterized in“binding to the same epitope as <CSF-1R>9D11.2E8 (deposit no. DSMACC2920)” has a percentage (%) of expected binding response of lowerthan 12 (% expected binding response <12).

The “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chain domains which are involved directly inbinding the antibody to the antigen. The variable light and heavy chaindomains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementary determining regions,CDRs). The framework regions adopt a β-sheet conformation and the CDRsmay form loops connecting the β-sheet structure. The CDRs in each chainare held in their three-dimensional structure by the framework regionsand form together with the CDRs from the other chain the antigen bindingsite. The antibody's heavy and light chain CDR3 regions play aparticularly important role in the binding specificity/affinity of theantibodies according to the invention and therefore provide a furtherobject of the invention.

The term “antigen-binding portion of an antibody” when used herein referto the amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially. CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy alpha-amino acids comprising alanine(three letter code: ala, one letter code: A), arginine (arg, R),asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C),glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine(his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K),methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine(ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y),and valine (val, V).

A further embodiment of the invention is a method for the production ofan antibody against human CSF-1R according to the inventioncharacterized in that the sequence of a nucleic acid encoding the heavychain of a human IgG1 class antibody binding to human CSF-1R and thenucleic acid encoding the light chain of said antibody are inserted intoan expression vector, said vector is inserted in a eukaryotic host cell,the encoded protein is expressed and recovered from the host cell or thesupernatant.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COScells, yeast, or E. coli cells, and the antibody is recovered from thecells (from the supernatant or after cells lysis).

Nucleic acid molecules encoding amino acid sequence variants ofanti-CSF-1R antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-CSF-1Rantibody.

The heavy and light chain variable domains according to the inventionare combined with sequences of promoter, translation initiation,constant region, 3′ untranslated region, polyadenylation, andtranscription termination to form expression vector constructs. Theheavy and light chain expression constructs can be combined into asingle vector, co-transfected, serially transfected, or separatelytransfected into host cells which are then fused to form a single hostcell expressing both chains.

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman. R. J., Mol. Biotechnol. 16(2000) 151-161: Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis, and others well known in the art. SeeAusubel, F., et al., eds. Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger. E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally. “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

As used herein, the expressions “cell”. “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibit various effector functions. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD),IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2.According to the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The Fc partof an antibody is directly involved in ADCC (antibody-dependentcell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity)based on complement activation, C1q binding and Fc receptor binding.Complement activation (CDC) is initiated by binding of complement factorC1q to the Fe part of most IgG antibody subclasses. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in the Fcpart. Such binding sites are known in the state of the art and describede.g. by Boackle, R. J., et al., Nature 282 (1979) 742-743. Lukas, T. J.,et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse, R, and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980)338-344, Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004,Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184, Hezareh, M.,et al., J. Virology 75 (2001) 12161-12168, Morgan. A., et al.,Immunology 86 (1995) 319-324, EP 0307434. Such binding sites are e.g.1234, L235, D270, N297, E318, K320, K322, P331 and P329 (numberingaccording to EU index of Kabat. E. A., see below). Antibodies ofsubclass IgG1, IgG2 and IgG3 usually show complement activation and C1qand C3 binding, whereas IgG4 do not activate the complement system anddo not bind C1q and C3.

In one embodiment the antibody according to the invention comprises a Fcpart derived from human origin and preferably all other parts of thehuman constant regions. As used herein the term “Fe part derived fromhuman origin” denotes a Fc part which is either a Fc part of a humanantibody of the subclass IgG1, IgG2, IgG3 or IgG4, preferably a Fc partfrom human IgG1 subclass, a mutated Fc part from human IgG1 subclass(preferably with a mutation on L234A+L235A), a Fc part from human IgG4subclass or a mutated Fc part from human IgG4 subclass (preferably witha mutation on S228P). Mostly preferred are the human heavy chainconstant regions of SEQ ID NO: 19 (human IgG1 subclass), SEQ ID NO: 20(human IgG1 subclass with mutations L234A and L235A), SEQ ID NO:21 humanIgG4 subclass), or SEQ ID NO:22 (human IgG4 subclass with mutationS228P).

In one embodiment the antibody according to the invention ischaracterized in that the constant chains are of human origin. Suchconstant chains are well known in the state of the art and e.g.described by Kabat. E. A., (see e.g. Johnson, G, and Wu, T. T., NucleicAcids Res. 28 (2000) 214-218). For example, a useful human heavy chainconstant region comprises an amino acid sequence of SEQ ID NO: 17. Forexample, a useful human light chain constant region comprises an aminoacid sequence of a kappa-light chain constant region of SEQ ID NO: 18.It is further preferred that the antibody is of mouse origin andcomprises the antibody variable sequence frame of a mouse antibodyaccording to Kabat.

The invention comprises a method for the treatment of a patient in needof therapy, characterized by administering to the patient atherapeutically effective amount of an antibody according to theinvention.

The invention comprises the use of an antibody according to theinvention for therapy.

Thus the antibodies according to the invention binding to the sameepitope were able to inhibit cell proliferation in CSF-1ligand-dependent and CSF-1 ligand independent cells. Especially theCSF-1R antibodies of the present invention are for use in the treatmentof CSF-1 ligand-dependent and CSF-1 ligand-independent CSF-1R mediateddiseases. This means that the CSF1-R mediated disease is eitherdependent of CSF-1 ligand and the corresponding signaling through CSF-1Rand/or independent of CSF-1 ligand and the corresponding signalingthrough CSF-1R. Signaling through CSF-1R is likely involved in tumorgrowth and metastasis.

One embodiment of the invention are the CSF-1R antibodies of the presentinvention for use in the treatment of “CSF-1R mediated diseases” or theCSF-1R antibodies of the present invention for use for the manufactureof a medicament in the treatment of “CSF-1R mediated diseases”, whichcan be described as follows:

There are 3 distinct mechanisms by which CSF-1R signaling is likelyinvolved in tumor growth and metastasis. The first is that expression ofCSF-ligand and receptor has been found in tumor cells originating in thefemale reproductive system (breast, ovarian, endometrium, cervical)(Scholl. S. M., et al., J. Natl. Cancer Inst. 86 (1994) 120-126;Kacinski, B. M., Mol. Reprod. Dev. 46 (1997) 71-74; Ngan. H. Y., et al.,Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N., et al., Cancer Res 67(2007) 1918-1926) and the expression has been associated with breastcancer xenograft growth as well as poor prognosis in breast cancerpatients. Two point mutations were seen in CSF-1R in about 10-20% ofacute myelocytic leukemia, chronic myelocytic leukemia andmyelodysplasia patients tested in one study, and one of mutations wasfound to disrupt receptor turnover (Ridge, S. A., et al., Proc. Natl.Acad. Sci USA 87 (1990) 1377-1380). However the incidence of themutations could not be confirmed in later studies (Abu-Duhier, F. M., etal., Br. J. Haematol. 120 (2003) 464-470). Mutations were also found insome cases of hepatocellular cancer (Yang, D. H., et al., HepatobiliaryPancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis(Abu-Duhier, F. M., et al., Br. J. Haematol. 120 (2003) 464-470).

Pigmented villonodular synovitis (PVNS) and Tenosynovial Giant celltumors (TGCT) can occur as a result of a translocation that fuses theM-CSF gene to a collagen gene COL6A3 and results in overexpression ofM-CSF (West, R. B., et al., Proc. Natl. Acad. Sci. USA 103 (2006)690-695). A landscape effect is proposed to be responsible fir theresulting tumor mass that consists of monocytic cells attracted by cellsthat express M-CSF. TGCTs are smaller tumors that can be relativelyeasily removed from fingers where they mostly occur. PVNS is moreaggressive as it can recur in large joints and is not as easilycontrolled surgically.

The second mechanism is based on blocking signaling through M-CSF/CSF-1Rat metastatic sites in bone which induces osteoclastogenesis, boneresorption and osteolytic bone lesions. Breast, multiple myeloma andlung cancers are examples of cancers that have been found to metastasizeto the bone and cause osteolytic bone disease resulting in skeletalcomplications. M-CSF released by tumor cells and stroma induces thedifferentiation of hematopoietic myeloid monocyte progenitors to matureosteoclasts in collaboration with the receptor activator of nuclearfactor kappa-B ligand-RANKL. During this process, M-CSF acts as apermissive factor by giving the survival signal to osteoclasts (Tanaka,S., et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1Ractivity during osteoclast differentiation and maturation with ananti-CSF-1R antibody is likely to prevent unbalanced activity ofosteoclasts that cause osteolytic disease and the associated skeletalrelated events in metastatic disease. Whereas breast, lung cancer andmultiple myeloma typically result in osteolytic lesions, metastasis tothe bone in prostate cancer initially has an osteoblastic appearance inwhich increased bone forming activity results in ‘woven bone’ which isdifferent from typical lamellar structure of normal bone. During diseaseprogression bone lesions display a significant osteolytic component aswell as high serum levels of bone resorption and suggests thatanti-resorptive therapy may be useful. Bisphosphonates have been shownto inhibit the formation of osteolytic lesions and reduced the number ofskeletal-related events only in men with hormone-refractory metastaticprostate cancer but at this point their effect on osteoblastic lesionsis controversial and bisphosphonates have not been beneficial inpreventing bone metastasis or hormone responsive prostate cancer todate. The effect of anti-resorptive agents in mixedosteolytic/osteoblastic prostate cancer is still being studied in theclinic (Choueiri. M. B., et al., Cancer Metastasis Rev. 25 (2006)601-609; Vessella, R. L. and Corey, E., Clin. Cancer Res. 12 (20 Pt 2)(2006) 6285s-6290s).

The third mechanism is based on the recent observation that tumorassociated macrophages (TAM) found in solid tumors of the breast,prostate, ovarian and cervical cancers correlated with poor prognosis(Bingle, L., et al., J. Pathol. 196 (2002) 254-265; Pollard, J. W., Nat.Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor byM-CSF and other chemokines. The macrophages can then contribute to tumorprogression through the secretion of angiogenic factors, proteases andother growth factors and cytokines and may be blocked by inhibition ofCSF-1R signaling. Recently it was shown by Zins et al (Zins, K., et al.,Cancer Res. 67 (2007) 1038-1045) that expression of siRNA of Tumornecrosis factor alpha (TNF alpha), M-CSF or the combination of bothwould reduce tumor growth in a mouse xenograft model between 34% and 50%after intratumoral injection of the respective siRNA. SiRNA targetingthe TNF alpha secreted by the human SW620 cells reduced mouse M-CSFlevels and led to reduction of macrophages in the tumor. In additiontreatment of MCF7 tumor xenografts with an antigen binding fragmentdirected against M-CSF did result in 40% tumor growth inhibition,reversed the resistance to chemotherapeutics and improved survival ofthe mice when given in combination with chemotherapeutics (Paulus, P., tal., Cancer Res. 66 (2006) 4349-4356).

TAMs are only one example of an emerging link between chronicinflammation and cancer. There is additional evidence for a link betweeninflammation and cancer as many chronic diseases are associated with anincreased risk of cancer, cancers arise at sites of chronicinflammation, chemical mediators of inflammation are found in manycancers; deletion of the cellular or chemical mediators of inflammationinhibits development of experimental cancers and long-term use ofanti-inflammatory agents reduce the risk of some cancers. A link tocancer exists for a number of inflammatory conditions among—those H.pylori induced gastritis for gastric cancer, Schistosomiasis for bladdercancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer andprostatitis for prostate cancer (Balkwill, F., et al., Cancer Cell 7(2005) 211-217). Macrophages are key cells in chronic inflammation andrespond differentially to their microenvironment. There are two types ofmacrophages that are considered extremes in a continuum of functionalstates: M1 macrophages are involved in Type 1 reactions. These reactionsinvolve the activation by microbial products and consequent killing ofpathogenic microorganisms that result in reactive oxygen intermediates.On the other end of the extreme are M2 macrophages involved in Type 2reactions that promote cell proliferation, tune inflammation andadaptive immunity and promote tissue remodeling, angiogenesis and repair(Mantovani, A., et al., Trends Immunol. 25 (2004) 677-686). Chronicinflammation resulting in established neoplasia is usually associatedwith M2 macrophages. A pivotal cytokine that mediates inflammatoryreactions is TNF alpha that true to its name can stimulate anti-tumorimmunity and hemorrhagic necrosis at high doses but has also recentlybeen found to be expressed by tumor cells and acting as a tumor promoter(Zins, K., et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F., CancerMetastasis Rev. 25 (2006) 409-416). The specific role of macrophageswith respect to the tumor still needs to be better understood includingthe potential spatial and temporal dependence on their function and therelevance to specific tumor types.

Thus one embodiment of the invention are the CSF-1R antibodies of thepresent invention for use in the treatment of cancer. The term “cancer”as used herein may be, for example, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva. Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma, lymphoma, lymphocytic leukemia, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers. Preferably such cancer is a breast cancer, ovariancancer, cervical cancer, lung cancer or prostate cancer. Preferably suchcancers are further characterized by CSF-1 or CSF-1R expression oroverexpression. One further embodiment the invention are the CSF-1Rantibodies of the present invention for use in the simultaneoustreatment of primary tumors and new metastases.

Thus another embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of periodontitis,histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone lossdue to cancer therapy, periprosthetic osteolysis, glucocorticoid-inducedosteoporosis, rheumatoid arthritis, psioratic arthritis, osteoarthritis,inflammatory arthridities, and inflammation.

Rabello, D., et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796has demonstrated that SNPs in the CSF1 gene exhibited a positiveassociation with aggressive periodontitis: an inflammatory disease ofthe periodontal tissues that causes tooth loss due to resorption of thealveolar bone.

Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is aproliferative disease of Langerhans dendritic cells that appear todifferentiate into osteoclasts in bone and extraosseous LCH lesions.Langerhans cells are derived from circulating monocytes. Increasedlevels of M-CSF that have been measured in sera and lesions where foundto correlate with disease severity (da Costa. C. E., et al., J. Exp.Med. 201 (2005) 687-693). The disease occurs primarily in a pediatricpatient population and has to be treated with chemotherapy when thedisease becomes systemic or is recurrent.

The pathophysiology of osteoporosis is mediated by loss of bone formingosteoblasts and increased osteoclast dependent bone resorption.Supporting data has been described by Cenci et al showing that ananti-M-CSF antibody injection preserves bone density and inhibits boneresorption in ovariectomized mice (Cenci. S., et al., J. Clin. Invest.105 (2000) 1279-1287). Recently a potential link between postmenopausalbone loss due to estrogen deficiency was identified and found that thepresence of TNF alpha producing T-cell affected bone metabolism (Roggia,C., et al., Minerva Med. 95 (2004) 125-132). A possible mechanism couldbe the induction of M-CSF by TNF alpha in vivo. An important role forM-CSF in TNF-alpha-induced osteoclastogenesis was confirmed by theeffect of an antibody directed against M-CSF that blocked the TNF alphainduced osteolysis in mice and thereby making inhibitors of CSF-1Rsignaling potential targets for inflammatory arthritis (Kitaura, H., etal., J. Clin. Invest. 115 (2005) 3418-3427).

Paget's disease of bone (PDB) is the second most common bone metabolismdisorder after osteoporosis in which focal abnormalities of increasedbone turnover lead to complications such as bone pain, deformity,pathological fractures and deafness. Mutations in four genes have beenidentified that regulate normal osteoclast function and predisposeindividuals to PDB and related disorders: insertion mutations inTNFRSF11A, which encodes receptor activator of nuclear factor (NF)kappaB (RANK)-a critical regulator of osteoclast function, inactivatingmutations of TNFRSF11B which encodes osteoprotegerin (a decoy receptorfor RANK ligand), mutations of the sequestosome 1 gene (SQSTM1), whichencodes an important scaffold protein in the NFkappaB pathway andmutations in the valosin-containing protein (VCP) gene. This geneencodes VCP, which has a role in targeting the inhibitor of NFkappaB fordegradation by the proteasome (Daroszewska, A, and Ralston, S. H., Nat.Clin. Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitorsprovide an opportunity to block the deregulation of the RANKL signalingindirectly and add an additional treatment option to the currently usedbisphosphonates.

Cancer therapy induced bone loss especially in breast and prostatecancer patients is an additional indication where a targeted CSF-1Rinhibitor could prevent bone loss (Lester, J. E., et al., Br. J. Cancer94 (2006) 30-35). With the improved prognosis for early breast cancerthe long-term consequences of the adjuvant therapies become moreimportant as some of the therapies including chemotherapy, irradiation,aromatase inhibitors and ovary ablation affect bone metabolism bydecreasing the bone mineral density, resulting in increased risk forosteoporosis and associated fractures (Lester, J. E., et al., Br. J.Cancer 94 (2006) 30-35). The equivalent to adjuvant aromatase inhibitortherapy in breast cancer is androgen ablation therapy in prostate cancerwhich leads to loss of bone mineral density and significantly increasesthe risk of osteoporosis-related fractures (Stoch, S. A., et al., J.Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

Targeted inhibition of CSF-1R signaling is likely to be beneficial inother indications as well when targeted cell types include osteoclastsand macrophages e.g. treatment of specific complications in response tojoint replacement as a consequence of rheumatoid arthritis. Implantfailure due to periprosthetic bone loss and consequent loosing ofprostheses is a major complication of joint replacement and requiresrepeated surgery with high socioeconomic burdens for the individualpatient and the health-care system. To date, there is no approved drugtherapy to prevent or inhibit periprosthetic osteolysis (Drees, P., etal., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).

Glucocorticoid-induced osteoporosis (GIOP) is another indication inwhich a CSF-1R inhibitor could prevent bone loss after longtermglucocorticocosteroid use that is given as a result of variousconditions among those chronic obstructive pulmonary disease, asthma andrheumatoid arthritis (Guzman-Clark, J. R., et al., Arthritis Rheum. 57(2007) 140-146; Feldstein, A. C., et al., Osteoporos. Int. 16 (2005)2168-2174).

Rheumatoid arthritis, psioratic arthritis and inflammatory arthriditiesare in itself potential indications for CSF-1R signaling inhibitors inthat they consist of a macrophage component and to a varying degree bonedestruction (Ritchlin, C. T., et al., J. Clin. Invest. 111 (2003)821-831). Osteoarthritis and rheumatoid arthritis are inflammatoryautoimmune disease caused by the accumulation of macrophages in theconnective tissue and infiltration of macrophages into the synovialfluid, which is at least partially mediated by M-CSF. Campbell, L K., etal., J. Leukoc. Biol. 68 (2000) 144-150, demonstrated that M-CSF isproduced by human-joint tissue cells (chondrocytes, synovialfibroblasts) in vitro and is found in synovial fluid of patients withrheumatoid arthritis, suggesting that it contributes to the synovialtissue proliferation and macrophage infiltration which is associatedwith the pathogenesis of the disease. Inhibition of CSF-1R signaling islikely to control the number of macrophages in the joint and alleviatethe pain from the associated bone destruction. In order to minimizeadverse effects and to further understand the impact of the CSF-1Rsignaling in these indications, one method is to specifically inhibitCSF-1R without targeting a myriad other kinases, such as Raf kinase.

Recent literature reports correlate increased circulating M-CSF withpoor prognosis and atherosclerotic progression in chronic coronaryartery disease (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000)655-665; Ikonomidis, I., et al., Eur. Heart. J. 26 (2005) p. 1618-1624);M-CSF influences the atherosclerotic process by aiding the formation offoam cells (macrophages with ingested oxidized LDL) that express CSF-1Rand represent the initial plaque (Murayama, T., et al., Circulation 99(1999) 1740-1746).

Expression and signaling of M-CSF and CSF-1R is found in activatedmicroglia. Microglia, which are resident macrophages of the centralnervous system, can be activated by various insults, including infectionand traumatic injury. M-CSF is considered a key regulator ofinflammatory responses in the brain and M-CSF levels increase in HIV-1,encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis asa consequence of autocrine signaling by M-CSF/CSF-1R results ininduction of inflammatory cytokines and nitric oxides being released asdemonstrated by e.g. using an experimental neuronal damage model (Hao,A. J., et al., Neuroscience 112 (2002) 889-900; Murphy, G. M., Jr., etal., J. Biol. Chem. 273 (1998) 20967-20971). Microglia that haveincreased expression of CSF-1R are found to surround plaques in AD andin the amyloid precursor protein V717F transgenic mouse model of AD(Murphy, G. M., Jr., et al., Am. J. Pathol. 157 (2000) 895-904). On theother hand op/op mice with fewer microglia in the brain resulted infibrilar deposition of A-beta and neuronal loss compared to normalcontrol suggesting that microglia do have a neuroprotective function inthe development of AD lacking in the op/op mice (Kaku, M., et al., BrainRes. Brain Res. Protoc. 12 (2003) 104-108).

Expression and signaling of M-CSF and CSF-1R is associated withinflammatory bowel disease (IBD) (WO 2005/046657). The term“inflammatory bowel disease” refers to serious, chronic disorders of theintestinal tract characterized by chronic inflammation at various sitesin the gastrointestinal tract, and specifically includes ulcerativecolitis (UC) and Crohn's disease.

-   The invention comprises the antibody characterized in comprising the    antibody binding to human CSF-1R being characterized by the above    mentioned epitope binding properties or alternatively by the above    mentioned amino acid sequences and amino acid sequence fragments for    the treatment of cancer.-   The invention comprises the antibody characterized in comprising the    antibody binding to human CSF-1R being characterized by the above    mentioned epitope binding properties or alternatively by the above    mentioned amino acid sequences and amino acid sequence fragments for    the treatment of bone loss.-   The invention comprises the antibody characterized in comprising the    antibody binding to human CSF-1R being characterized by the above    mentioned epitope binding properties or alternatively by the above    mentioned amino acid sequences and amino acid sequence fragments for    the prevention or treatment of metastasis.-   The invention comprises the antibody characterized in comprising the    antibody binding to human CSF-1R being characterized by the above    mentioned epitope binding properties or alternatively by the above    mentioned amino acid sequences and amino acid sequence fragments for    treatment of inflammatory diseases.-   The invention comprises the use of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for the treatment of cancer or alternatively for the    manufacture of a medicament for the treatment of cancer.-   The invention comprises the use of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for the treatment of bone loss or alternatively for the    manufacture of a medicament for the treatment of bone loss.-   The invention comprises the use of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for the prevention or treatment of metastasis or    alternatively for the manufacture of a medicament for the prevention    or treatment of metastasis.-   The invention comprises the use of an antibody characterized in    comprising the antibody binding to human CSF-1R being characterized    by the above mentioned epitope binding properties or alternatively    by the above mentioned amino acid sequences and amino acid sequence    fragments for treatment of inflammatory diseases or alternatively    for the manufacture of a medicament for the treatment of    inflammatory diseases.-   In one embodiment the antibodies according to the invention have one    or more of the following properties    -   a) inhibition of the growth of NIH3T3—wildtype CSF-1R        recombinant cells by 90% or more at an antibody concentration of        10 μg/ml (see Example 2);    -   b) inhibition of the growth of NIH3T3—mutant CSF-1R L301S Y969F        recombinant cells by 60% or more at an antibody concentration of        10 μg/ml ((e.g. by see Example 2);    -   c) inhibition of the CSF-1/CSF-1R interaction (e.g. with an IC50        value of 15 ng/ml or lower, see Example 3);    -   d) Inhibition of CSF-1-induced CSF-1R phosphorylation in        wildtype NIH3T3-CSF-1R recombinant cells (e.g. with an IC50        value of 80 ng/ml or lower, see Example 4);    -   e) inhibition of the growth of BeWo tumor cells (e.g. by 80% or        more at an antibody concentration of 10 μg/ml see Example 7);    -   f) inhibition of macrophage differentiation (e.g. with an IC50        value of 0.8 nM or lower, see Example 8).-   The invention comprises in one aspect an antibody binding to human    CSF-1R, wherein the antibody binds to the same epitope as the    deposited antibody DSM ACC2920 and wherein the antibody has one or    more of the following properties:    -   a) inhibition of the growth of NIH3T3—wildtype CSF-1R        recombinant cells by 90% or more at an antibody concentration of        10 μg/ml (see Example 2);    -   b) inhibition of the growth of NIH3T3—mutant CSF-1R L301S Y969F        recombinant cells by 60% or more at an antibody concentration of        10 μg/ml ((e.g. by see Example 2);    -   c) inhibition of the CSF-1/CSF-1R interaction (e.g. with an IC50        value of 15 ng/ml or lower, see Example 3);    -   d) Inhibition of CSF-1-induced CSF-1R phosphorylation in        wildtype NIH13T3-CSF-1R recombinant cells (e.g. with an IC50        value of 80 ng/ml or lower, see Example 4);    -   e) inhibition of the growth of BeWo tumor cells (e.g. by 80% or        more at an antibody concentration of 10 μg/ml see Example 7);    -   f) inhibition of macrophage differentiation (e.g. with an IC50        value of 0.8 nM or lower, see Example 8).

In another aspect, the present invention provides a composition, e.g. apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption/resorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor injection or infusion.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the preparation of sterileinjectable solutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. In addition towater, the carrier can be, for example, an isotonic buffered salinesolution.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient (effectiveamount). The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The invention comprises the use of the antibodies according to theinvention for the treatment of a patient suffering from cancer,especially from colon, lung or pancreas cancer.

The invention comprises also a method for the treatment of a patientsuffering from such disease.

The invention further provides a method for the manufacture of apharmaceutical composition comprising an effective amount of an antibodyaccording to the invention together with a pharmaceutically acceptablecarrier and the use of the antibody according to the invention for sucha method.

The invention further provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

The invention also provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

The following examples and sequence listing are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Antibody Deposition

The preferred hybridoma cell line according to the invention, hybridomacell line <CSF-1R>9D11.2E was deposited, under the Budapest Treaty onthe international recognition of the deposit of microorganisms for thepurposes of patent procedure, with Deutsche Sammlung von Mikroorganismenund Zdelkulturen GmbH (DSMZ), Germany, on Jun. 10, 2008 under AccessionNo. DSM ACC 2920.

Cell line Deposition No. Date of Deposit <CSF-1R>9D11.2E8 DSM ACC2920Oct. 6, 2008

The antibodies obtainable from said cell line are preferred embodimentsof the invention.

Description of the Sequences

SEQ ID NO: 1 heavy chain CDR3, <CSF-1R>9D11.2E8 SEQ ID NO: 2 heavy chainCDR2, <CSF-1R>9D11.2E8 SEQ ID NO: 3 heavy chain CDR1, <CSF-1R>9D11.2E8SEQ ID NO: 4 light chain CDR3, <CSF-1R>9D11.2E8 SEQ ID NO: 5 light chainCDR2, <CSF-1R>9D11.2E8 SEQ ID NO: 6 light chain CDR1, <CSF-1R>9D11.2E8SEQ ID NO: 7 heavy chain variable domain, <CSF-1R>9D11.2E8 SEQ ID NO: 8light chain variable domain, <CSF-1R>9D11.2E8 SEQ ID NO: 9 heavy chainCDR3, <CSF-1R>10H2.2F12 SEQ ID NO: 10 heavy chain CDR2,<CSF-1R>10H2.2F12 SEQ ID NO: 11 heavy chain CDR1, <CSF-1R>10H2.2F12 SEQID NO: 12 light chain CDR3, <CSF-1R>10H2.2F12 SEQ ID NO: 13 light chainCDR2, <CSF-1R>10H2.2F12 SEQ ID NO: 14 light chain CDR1,<CSF-1R>10H2.2F12 SEQ ID NO: 15 heavy chain variable domain,<CSF-1R>10H2.2F12 SEQ ID NO: 16 light chain variable domain,<CSF-1R>10H2.2F12 SEQ ID NO: 17 γ1 heavy chain constant region SEQ IDNO: 18 κ light chain constant region SEQ ID NO: 19 human heavy chainconstant region derived from IgG1 SEQ ID NO: 20 human heavy chainconstant region derived from IgG1 mutated on L234A and L235A SEQ ID NO:21 human heavy chain constant region derived from IgG4 SEQ ID NO: 22human heavy chain constant region derived from IgG4 mutated onS228P SEQID NO: 23 wildtype CSF-1R (wt CSF-1R) SEQ ID NO: 24 mutant CSF-1R L301SY969F

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Example 1 Generation of a Hybridoma Cell Line Producing Anti-CSF-1RAntibodies Immunization Procedure of NMRI Mice

NMRI mice were immunized with an expression vector pDisplay™(Invitrogen, USA) encoding the extracellular domain of huCSF-1R byutilizing electroporation. Every mouse was 4 times immunized with 100 μgDNA. When serum titers of anti-huCSF-1R were found to be sufficient,mice were additionally boosted once with 50 μg of a 1:1 mixture huCSF-1RECD/huCSF-1R ECDhuFc chimera in 200 μl PBS intravenously (i.v.) 4 and 3days before fusion.

Antigen Specific ELISA

Anti-CSF-1R titers in sera of immunized mice were determined by antigenspecific ELISA.

0.3 μg/ml huCSF-1R-huFc chimera (soluble extracellular domain) wascaptured on a streptavidin plate (MaxiSorb; MicroCoat, DE, Cat.No.11974998/MC1099) with 0.1 mg/ml biotinylated anti Fcγ (JacksonImmunoResearch, Cat.No. 109-066-098) and horse radish peroxidase(HRP)-conjugated F(ab′)₂ anti mouse IgG (GE Healthcare, UK, Cat.No.NA9310V) diluted 1/800 in PBS/0.05% Tween20/0.5% BSA was added. Serafrom all taps were diluted 1/40 in PBS/0.05% Tween20/0.5% BSA andserially diluted up to 1/1638400. Diluted sera were added to the wells.

Pre-tap serum was used as negative control. A dilution series of mouseanti-human CSF-1R Mab3291 (R&D Systems, UK) from 500 ng/ml to 0.25 ng/mlwas used as positive control. All components were incubated together for1.5 hours. Wells were washed 6 times with PBST (PBS/0.2% Tween20) andassays were developed with freshly prepared ABTS® solution (1 mg/ml)(ABTS: 2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 10minutes at RT. Absorbance was measured at 405 nm.

Hybridoma Generation

The mouse lymphocytes can be isolated and fused with a mouse myelomacell line using PEG based standard protocols to generate hybridomas. Theresulting hybridomas are then screened for the production ofantigen-specific antibodies. For example, single cell suspensions ofsplenic derived lymphocytes from immunized mice are fused to Ag8non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50%PEG. Cells are plated at approximately 10⁴ in flat bottom 96 well microtiter plate, followed by about two weeks incubation in selective medium.Individual wells are then screened by ELISA for human anti-CSF-1Rmonoclonal IgM and IgG antibodies. Once extensive hybridoma growthoccurs, the antibody secreting hybridomas are replated, screened again,and if still positive for human IgG, anti-CSF-1R monoclonal antibodies,can be subcloned by FACS. The stable subclones are then cultured invitro to produce antibody in tissue culture medium for characterization.

Culture of Hybridomas

Generated muMAb hybridomas were cultured in RPMI 1640 (PAN—Catalogue No.(Cat. No.) PO4-17500) supplemented with 2 mM L-glutamine (GIBCO—Cat. No.35050-038), 1 mM Na-Pyruvat (GIBCO—Cat. No. 11360-039), 1×NEAA(GIBCO—Cat. No. 11140-035), 10% FCS (PAA—Cat. No. A15-649), 1× Pen Strep(Roche—Cat. No. 1074440), 1× Nutridoma CS (Roche—Cat. No. 1363743), 50μM Mercaptoethanol (GIBCO—Cat. No. 31350-010) and 50 U/ml IL 6 mouse(Roche—Cat. No. 1 444 581) at 37° C., and 5% CO₂.

Example 2

Selection of Antibodies Via Growth Inhibition of NIH3T3-CSF-1R (WildtypeCSF-1R or Mutant CSF-1R L301S Y969F) Recombinant Cells in 3D CultureUnder Treatment with Anti-CSF-1R Monoclonal Antibodies(CellTiterGlo-Assay)

NIH 3T3 cells (ATCC No. CRL-2795), retrovirally infected with either anexpression vector for full-length wildtype CSF-1R (SEQ ID NO: 23) ormutant CSF-1R L301S Y969F (SEQ ID NO: 24), were cultured in DMEM highglucose media (PAA, Pasching, Austria) supplemented with 2 mML-glutamine, 2 mM sodium pyruvate and non-essential amino acids and 10%fetal bovine serum (Sigma, Taufkirchen, Germany) on poly-HEMA(poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, Pa. USA))coated dishes to prevent adherence to the plastic surface. Cells areseeded in medium replacing serum with 5 ng/ml sodium selenite, 10 mg/mltransferrin, 400 μg/m BSA and 0.05 mM 2-mercaptoethanol. When treatedwith 100 ng/ml huCSF-1 (Biomol, Hamburg, Germany) wtCSF-1R expressingcells form dense spheroids that grow three dimensionally, a propertythat is called anchorage independence. These spheroids resemble closelythe three dimensional architecture and organization of solid tumors insitu.

Mutant CSF-1R recombinant cells are able to form spheroids independentof the CSF-1 ligand. Spheroid cultures were incubated for 3 days in thepresence of 10 μg/ml antibody. The CellTiterGlo assay was used to detectcell viability by measuring the ATP-content of the cells.

TABLE 1a Survival of cells expressing wildtype or mutant CSF-1R undertreatment with CSF-1R antibodies wtCSF-1R Mutant CSF-1R Clone % survival% survival <CSF-1R>9D11.2E8 7 33 <CSF-1R>10H2.2F12 3 35 SC2-4A5  62**  66*** **average of 15 different experiments, ***average of 6 differentexperiments

TABLE 1b Inhibition of the growth of NIH3T3 - wildtype CSF-1R or mutantCSF-1R L301S Y969F recombinant cells wtCSF-1R Mutant CSF-1R Clone %Inhibition % Inhibition <CSF-1R>9D11.2E8 93 67 <CSF-1R>10H2.2F12 97 65SC2-4A5  38**   34*** **average of 15 different experiments, ***averageof 6 different experiments

Thus the antibodies according to the invention binding to the sameepitope were able to inhibit cell proliferation in CSF-1ligand-dependent and/or CSF-1 ligand independent cells.

In a further experiment the anti-CSF-1R antibodies 1.2.SM1.19 (liganddisplacing CSF-1R antibody described in WO 2009/026303), CXIIG6 (liganddisplacing CSF-1R antibody described in WO 2009/1112245), the goatpolyclonal anti-CSF-1R antibody ab10676 (abeam), were investigated fortheir ability to inhibit the growth of NIH3T3—mutant CSF-1R L301S Y969F.Spheroid cultures were incubated for 3 days in the presence of differentconcentrations of antibody in order to determine an IC30 (concentrationwith 30 percent inhibition of cell viability). Maximum concentration was20 μg/ml The CellTiterGlo assay was used to detect cell viability bymeasuring the ATP-content of the cells.

For all three CSF-1R antibodies 1.2.SM1.19, CXIIG6, and ab10676 thepercentage of inhibition the growth of NIH3T3—mutant CSF-1R L301S Y969Frecombinant cells was 0% percent or even lower event at the highestconcentration of 20 μg/ml (which means that 1.2.SM1.19 and CXIIG6 werenot only unable to inhibit the cell growth of such NIH3T3—mutant CSF-1RL301S Y969F recombinant cells, instead they even stimulated the growthof such cells (1.2.SM1.19 showed 19% stimulation at 20 μg/ml and CXIIG6showed 36% stimulation at 20 μg/ml)

Example 3 Inhibition of CSF-1/CSF-1R Interaction (ELISA)

The test was performed on 384 well microtiter plates (MicroCoat, DE,Cat.No. 464718) at RT. After each incubation step plates were washed 3times with PBST.

At the beginning, plates were coated with 0.5 mg/ml goat F(ab′)₂biotinylated anti Fcγ (Jackson ImmunoResearch, Cat.No. 109-006-170) for1 hour (h).

Thereafter the wells were blocked with PBS supplemented with 0.2%Tween®-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 0.5 h. 75 ng/ml ofhuCSF-1R-huFc chimera (soluble extracellular domain) was immobilized toplate for 1 h. Then dilutions of purified antibodies in PBS/0.05%Tween20/0.5% BSA were incubated for 1 h. After adding a mixture of 3ng/ml CSF-1 (Biomol, DE, Cat.No. 60530), 50 ng/ml biotinylated antiCSF-1 clone BAF216 (R&D Systems, UK) and 1:5000 diluted streptavidin HRP(Roche Diagnostics GmbH, DE, Cat.No. 11089153001) for 1 h the plateswere washed 6 times with PBST. Anti CSF-1R SC-02, clone 2-4A5 (SantaCruz Biotechnology, US), which inhibits the ligand-receptor interaction,was used as positive control. Plates were developed with freshlyprepared BM Blue® POD substrate solution (BM Blue®:3,3′-5,5′-Tetramethylbenzidine, Roche Diagnostics GmbH, DE, Cat.No.11484281001) for 30 minutes at RT. Absorbance was measured at 370 nm.All anti-CSF-1R antibodies showed significant inhibition of the CSF-1binding to CSF-1R (see Table 2). Anti CSF-1R SC-02, clone 2-4A5 (SantaCruz Biotechnology. US), which inhibits the ligand-receptor interaction,was used as reference control.

TABLE 2 Calculated IC50 values for the inhibition of the CSF-1/CSF-1Rinteraction IC50 CSF-1/CSF-1R Clone Inhibition [ng/ml] <CSF-1R>9D11.2E810.0 SC-2-4A5 30.9

Example 4 Inhibition of CSF-1-Induced CSF-1R Phosphorylation inNIH3T3-CSF-1R Recombinant Cells

4.5×10³ NIH 3T3 cells, retrovirally infected with an expression vectorfor full-length CSF-1R (SEQ ID NO: 23), were cultured in DMEM (PAA Cat.No. E15-011), 2 mM L-glutamine (Sigma, Cat.No. G7513, 2 mM Sodiumpyruvate, 1× nonessential aminoacids, 10% FKS (PAA, Cat.No. A15-649) and100 μg/ml PenStrep (Sigma, Cat.No. P4333 [10 mg/ml]) until they reachedconfluency. Thereafter cells were washed with serum-free DMEM media (PAACat.No. E15-011) supplemented with sodium selenite [5 ng/ml] (Sigma.Cat.No. S9133), transferrin [10 μg/ml] (Sigma, Cat.No. T8158), BSA [400μg/ml] (Roche Diagnostics GmbH, Cat.No. 10735078), 4 mM L-glutamine(Sigma, Cat.No. G7513), 2 mM sodium pyruvate (Gibco, Cat.No. 11360), 1×nonessential aminoacids (Gibco, Cat: 11140-035), 2-mercaptoethanol [0.05mM] (Merck, Cat.No. M7522), 100 μg/ml and PenStrep (Sigma, Cat. No.P4333) and incubated in 30 μl of the same medium for 16 hours to allowfor receptor up-regulation. 10 μl of diluted anti-CSR-1R antibodies wereadded to the cells for 1.5 h. Then cells were stimulated with 10 μl of100 ng/ml huM-CSF-1 (Biomol Cat.No. 60530) for 5 min. After theincubation, supernatant was removed, cells were washed twice with 80 μlof ice-cold PBS and 50 μl of freshly prepared ice-cold lysis buffer (150mM NaCl/20 mM Tris pH 7.5/1 mM EDTA/1 mM EGTA/1% Triton X-100/1 proteaseinhibitor tablet (Roche Diagnostics GmbH Cat.No. 1 836 170) per 10 mlbuffer; 10 μl/ml phosphatase inhibitor cocktail 1 (Sigma Cat.No.P1-2850, 100× Stock); 10 μl/ml protease inhibitor 1 (Sigma Cat.No.P-5726, 100× Stock)/10 μl/ml 1 M NaF) was added. After 30 minutes on icethe plates were shaken vigorously on a plateshaker for 3 minutes andthen centrifuged 10 minutes at 2200 rpm (Heracus Megafuge 10).

The presence of phosphorylated and total CSF-1 receptor in the celllysate was analyzed with Elisa. For detection of the phosphorylatedreceptor the kit from R&D Systems (Cat. No. DYC3268-2) was usedaccording to the instructions of the supplier. For detection of totalCSF-1R 10 μl of the lysate was immobilized on plate by use of thecapture antibody contained in the kit. Thereafter 1:750 dilutedbiotinylated anti CSF-1R antibody BAF329 (R&D Systems) and 1:1000diluted streptavidin-HRP conjugate was added. After 60 minutes plateswere developed with freshly prepared ABTS® solution and the absorbancewas detected. Data were calculated as % of positive control withoutantibody and the ratio value phospho/total receptor expressed. Thenegative control was defined without addition of M-CSF-1. Anti CSF-1RSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C. J.,et al., Cell 41 (1985) 665-676), which inhibits the ligand-receptorinteraction, was used as reference control.

TABLE 3 Calculated IC₅₀ values for the inhibition of CSF-1 receptorphosphorylation. IC50 CSF-1R Phosphorylation Clone [ng/ml]<CSF-1R>9D11.2E8 72.8 <CSF-1R>10H2.2F12 44.5 SC-2-4A5 412.0

Example 5 Determination of the Affinity of Anti-CSF-1R Antibodies toCSF-1R

Instrument: BIACORE® A100

Chip: CMS (Biacore BR-1006-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1006-72), pH 7.4, 35° C.

For affinity measurements 36 μg/ml anti mouse Fcγ antibodies (from goat,Jackson Immuno Research JIR115-005-071) have been coupled to the chipsurface for capturing the antibodies against CSF-1R. CSF-1R ECD(R&D-Systems 329-MR or in-house subclonedpCMV-presS-HisAvitag-hCSF-1R-ECD were added in various concentrations insolution. Association was measured by an CSF-1R-injection of 1.5 minutesat 35° C.; dissociation was measured by washing the chip surface withbuffer fir 10 minutes at 35° C. Anti CSF-1R SC-02, clone 2-4A5 (SantaCruz Biotechnology, US; see also Sherr, C. J., et al., Cell 41 (1985)665-676), which inhibits the ligand-receptor interaction, was used asreference control.

For calculation of kinetic parameters the Langmuir 1:1 model was used.

TABLE 4 Affinity data measured by SPR (BIACORE ® A100) at 35° C. K_(D)Clone (nM) k_(a) (1/Ms) k_(d) (1/s) t_(1/2) (min) <CSF-1R>9D11.2E8 0.774.7E+05 3.6E−04 32.00 <CSF-1R>10H2.2F12 0.78 5.2E+05 4.0E−04 28.60SC-2-4A5 2.73 5.09E+05  1.39E−03  8.31

Example 6 Epitope Mapping of Anti-CSF-1R Monoclonal Antibodies Based onCross-Competition by Utilizing SPR

Instrument: BIACORE® A100

Chip: CMS (Biacore BR-1006-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1006-72). pH 7.4, 25° C.

For epitope mapping assays via cross-competition 36 μg/ml anti mouse Fcγantibodies or anti rat Fcγ antibodies (from goat, Jackson ImmunoResearch Cat.No. 115-005-071 and Cat. No. 112-005-071) have been coupledto sensor chip surface for presentation of the antibody against CSF-1R.After capture from 5 μg/mm anti-CSF-1R monoclonal antibodies freebinding capacities of capture antibodies have been blocked with 250μg/ml mouse or rat immunoglobulins (Pierce Cat. No. 31202 and PierceCat. No. 31233), followed by injection of 12.5 μg/ml CSF-1R (R&D-SystemsCat.No. 329-MR) for 2 min. Binding of second anti-CSF-1R antibody hasbeen analyzed by injection for 2 min, dissociation was measured bywashing with buffer for 5 minutes. The assay and the measurements wereconducted at 25° C. The specific binding of the second anti-CSF-1Rantibody has been referenced against spot with the same chip setup upbut only without injection of CSF-1R. The cross competition data havebeen calculated in percentage (%) of expected binding response of thesecond anti-CSF-1R antibody. The item “percentage (%) of expectedbinding response” for binding of the second antibody was calculated by“100*relative Response(general_stability_early)/rMax”, where rMax iscalculated by “relative Response(general_stability_late)*antibodymolecular weight/antigen molecular weight” as described in Biacoreepitope mapping instructions (for BIACORE® A100 instrument).

The minimal binding response was also calculated from the pairs ofidentical antibody 1 and 2. Thereof the obtained maximal value+50% wasset as threshold for significant binding competition (see table X e.g.for antibody <CSF-1R>9D11.2E8 calculated threshold is 8+4=12). Thus an“anti-CSF-1R antibody binding to the same epitope as <CSF-1R>9D11.2E8”has a percentage (%) of expected binding response <12.

The anti-CSF-1R SC-02, clone 2-4A5 (Santa Cruz Biotechnology, US, seealso Sherr, C. J., et al., Cell 41 (1985) 665-676), which inhibit theligand-receptor interaction, was used as reference control.

TABLE 5 The epitope mapping via cross-competition data of anti CSF-1Rantibodies Antibody 2 <CSF-1R> <CSF-1R> SC-02, clone Antibody 1 9D11.2E810H2.2F12 2-4A5 <CSF-1R> 8 10 37 9D11.2E8 <CSF-1R> −1 1 42 10H2.2F12SC-02, clone 48 53 −2 2-4A5

The results indicate that the antibodies <CSF-1R>9D11.2E8, and<CSF-1R>10H2.2F12 all bind to the same epitope, while e.g. SC-2-4A5binds to another epitope and does not crossreact (crosscompete forbinding) with the antibodies according to the invention.

Also anti-CSF-1R antibodies Mab 2F11, 2E10, 2H7 and 1G10 fromWO2011070024 (which are able to inhibit the growth of NIH3T3—wildtypeCSF-1R or mutant CSF-1R L301S Y969F recombinant cells) were tested withantibodies of the present invention in a further experiments, whether tobind to the same epitope. These epitope mapping (via-cross-competition)results clearly indicated that the antibodies <CSF-1R>9D11.2E8, and<CSF-1R>10H2.2F12 bind to a different epitope than Mab 2F11, 2E10, 2H7and 1G10 from WO2011070024.

In a further separate experiment, which carried analogously as describedin WO2011070024 in Example 10, it was determined whether antibodies<CSF-1R>9D11.2E8, and <CSF-1R>10H2.2F12 bind to domains D1-D3 of theCSF1R Extracellular domain (CSF-1R-ECD) (D11-D3). This determinationresulted in the finding that antibodies <CSF-1R>9D11.2E8, and<CSF-1R>10H2.2F12 do not bind to CSF-1R ECD (D1-D3). Thus the antibodies<CSF-1R>9D11.2E8, and <CSF-1R>10H2.2F12 bind to human CSF-1RExtracellular Domain (comprising domains D1 to D5) and do not bind todomains D1 to D3 of the extracellular domain of human CSF-1R.Consequently antibodies <CSF-1R>9D11.2E8, and <CSF-1R>10H2.2F12 bind tothe (dimerization) domains D4 to D5 of the extracellular domain of humanCSF-1R.

Example 7

Growth Inhibition of BeWo Tumor Cells in 3D Culture Under Treatment withAnti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K media(Sigma, Steinheim, Germany) supplemented with 10%/FBS (Sigma) and 2 mML-glutamine. 5×10⁴ cells/well were seeded in 96-well poly-HEMA(poly(2-hydroxyethylmethacrylate)) coated plates containing F12K mediumsupplemented with 0.5% FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1and 10 μg/ml of different anti-CSF-1R monoclonal antibodies were addedand incubated for 6 days. The CellTiterGlo assay was used to detect cellviability by measuring the ATP-content of the cells in relative lightunits (RLU). When BeWo spheroid cultures were treated with differentanti-CSF-1R antibodies (10 μg/ml) inhibition of CSF-1 induced growth wasobserved. To calculate antibody-mediated inhibition the mean RLU valueof unstimulated BeWo cells was subtracted from all samples. Mean RLUvalue of CSF-1 stimulated cells was set arbitrarily to 100%. Mean RLUvalues of cells stimulated with CSF-1 and treated with anti-CSF-1Rantibodies were calculated in % of CSF-1 stimulated RLUs. Table 6 showsthe calculated data; FIG. 1 depicts mean RLU values. Each mean value wasderived from triplicates.

TABLE 6 % inhibition 10 μg/ml Clone antibody concentration CSF-1 only 0<CSF-1R>9D11.2E8 88 <CSF-1R>10H2.2F12 100 SC-02, clone 2-4A5 40

Example 8

Inhibition of Macrophage Differentiation Under Treatment withAnti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

Monocytes isolated from peripheral blood using the RosetteSep™ HumanMonocyte Enrichment Cocktail (StemCell Tech.—Cat. No. 15028). Enrichedmonocyte populations were seeded into 96 well microtiterplates (2.5×10⁴cells/well) in 100 μl RPMI 1640 (Gibco—Cat. No. 31870) supplemented with10 FCS (GIBCO—Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO—Cat. No.25030) and 1× PenStrep (Roche Cat. No. 1 074 440) at 37′C and 5% CO₂.When 150 ng/ml huCSF-1 was added to the medium, a clear differentiationinto adherent macrophages could be observed. This differentiation couldbe inhibited by addition of anti-CSF-1R antibodies. Furthermore, themonocyte survival is affected and could be analyzed by CellTiterGlo(CTG) analysis. From the concentration dependent inhibition of thesurvival of monocytes by antibody treatment, an IC₅₀ was calculated (seeTable 7).

TABLE 7 Clone IC₅₀ [nM] <CSF-1R>9D11.2E8 0.7 <CSF-1R>10H2.2F12 0.6SC-02, clone 2-4A5 2.4

In a separate experiment cynomolgous monkey derived monocytes isolatedfrom peripheral blood by Ficoll separation followed by magnetic sortingfor CD14 (Miltenyi Biotec Cat.No. 130091097) were tested under identicalassay conditions at 5 μg/ml antibody concentration each.<CSF-1R>9D11.2E8 showed 33% inhibition, <CSF-1R>10H12.2F12 18%inhibition. (In contrast Anti-CSF-1R Mab 2F11 described in WO2011070024(A1) inhibited the survival of monkey monocytes by 99%).

1-14. (canceled)
 15. An isolated nucleic acid encoding an antibodybinding to CSF-1R, wherein the antibody comprises a heavy chain variabledomain and alight chain variable domain, and wherein: a) the heavy chainvariable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 regionof SEQ ID NO: 2, and a CDR1 region of SEQ ID NO: 3, and the light chainvariable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 regionof SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6, or b) the heavy chainvariable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 regionof SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the lightchain variable domain comprises a CDR3 region of SEQ ID NO: 12, a CDR2region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO:
 14. 16. Anexpression vector comprising the nucleic acid of claim 15 for theexpression of the antibody in a prokaryotic or eukaryotic host cell. 17.A prokaryotic or eukaryotic host cell comprising the vector of claim 16.18. A method for the production of a recombinant antibody, the methodcomprising expressing the nucleic acid of claim 15 in a prokaryotic oreukaryotic host cell and recovering the antibody from said cell or cellculture supernatant. 19-21. (canceled)
 22. The isolated nucleic acid ofclaim 15, wherein the heavy chain variable domain comprising a CDR3region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 regionof SEQ ID NO: 3, and the light chain variable domain comprising a CDR3region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a CDR1 regionof SEQ ID NO:6.
 23. An expression vector comprising the nucleic acid ofclaim 22 for the expression of the antibody in a prokaryotic oreukaryotic host cell.
 24. A prokaryotic or eukaryotic host cellcomprising the vector of claim
 23. 25. A method for the production of arecombinant antibody, the method comprising expressing the nucleic acidof claim 22 in a prokaryotic or eukaryotic host cell and recovering theantibody from said cell or cell culture supernatant.
 26. The isolatednucleic acid of claim 15, wherein the heavy chain variable domaincomprising a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO:10, and a CDR1 region of SEQ ID NO: 11, and the light chain variabledomain comprising a CDR3 region of SEQ ID NO: 12, a CDR2 region of SEQID NO: 13, and a CDR1 region of SEQ ID NO:
 14. 27. An expression vectorcomprising the nucleic acid of claim 26 for the expression of theantibody in a prokaryotic or eukaryotic host cell.
 28. A prokaryotic oreukaryotic host cell comprising the vector of claim
 27. 29. A method forthe production of a recombinant antibody, the method comprisingexpressing the nucleic acid of claim 26 in a prokaryotic or eukaryotichost cell and recovering the antibody from said cell or cell culturesupernatant.
 30. The isolated nucleic acid of claim 15, wherein theantibody is of human IgG4 subclass or is of human IgG1 subclass.
 31. Theisolated nucleic acid of claim 15, wherein the antibody is a CDRgrafted, humanized, T cell epitope depleted, chimeric, single chain, ormultispecific antibody.
 32. The isolated nucleic acid of claim 15,wherein the antibody is an antigen binding fragment.